The Dkk1 gene, also called Dickkopf-1 (German for “big head” and “stubborn”), is a prototype member of a gene family that encodes secreted glycoproteins. These proteins control cell fate and neural patterning during embryonic development. The other Dkk family members include Dkk-2, Dkk-3 and Dkk-4. All four Dkk proteins are secreted and are synthesized as precursor proteins with an N-terminal signal peptide and two conserved cysteine-rich domains, which are separated by a linker region.
Evidence has been presented showing that Dkk1 functions as a potent inhibitor of the Wnt signaling pathway, a pathway that enables appropriate positioning and development of the embryonic brain and other organ structures (Shou et al., 2002 Oncogene 21: 878-889). Dkk-4 functions as another Wnt antagonist. Dkk-3 has not been demonstrated to affect Wnt signaling, and Dkk-2 acts as both an antagonist and an agonist of Wnt signaling, depending on the cellular context in which it is found.
The Wnt pathway consists of many highly conserved Wnt protein ligands that bind to the Frizzled family of receptors and trigger a cytoplasmic signal transduction cascade in which glycogen synthase kinase-3 (GSK-3) and β-catenin play essential roles. In the absence of Wnt signaling, β-catenin is associated with a cytoplasmic complex containing GSK-3, axin and the adenomatous polyposis coli protein (APC). In this complex, GSK-3 constitutively phosphorylates β-catenin. The phosphorylation of β-catenin permits it to become ubiquinated. The ubiquitin moiety marks the protein for subsequent degradation. In the presence of Wnt signaling, GSK-3 is inactivated, leading to free and unphosphorylated β-catenin building up in the cytoplasm. This β-catenin pool then translocates to the nucleus, where it associates with members of the lymphocyte enhancer factor (LEF)/TCF family of transcription factors to activate a variety of target genes involved in growth, development and oncogenesis.
Some of the Wnt target genes include cyclin D1, engrailed-2, cyclooxygenase-2, c-myc, and numerous metalloproteinases. The homeostasis of the Wnt pathway is maintained in part by Wnt antagonists and agonists. Thus, the role of Dkk1, a Wnt antagonist, would serve to diminish the accumulation of free β-catenin. The balance of β-catenin pools in the cytoplasm and in the nucleus determines “in part”, the outcome of Wnt signaling (Shou et al., 2002). The Dkk proteins (especially Dkk1) therefore play an active role in Wnt signaling. Dkk1 has been shown to be a target for the p53 tumor suppressor (Shou et al., 2002).
Therefore, adequate amounts of research grade Dkk1 or other Dkk proteins are needed for further study of p53 and Wnt signaling and their relationship to cancer and development.
Dkk1 has been produced via transient transfections with culture media being used as the protein is released into the culture media. Commercial vendors of Dkk1 also exist, such as R&D Systems (Minneapolis, Minn.). However, the commercially available forms of Dkk1 are in small quantities and costly (10 μg/$ 300); the commercially available forms also may not be functional depending on the assay used, if at all. Additionally, the commercially available form of Dkk1, as well as other forms taught in the literature, are either produced in low quantities, at high cost, were never purified, have reduced or altered biological activities or a combination of these flaws. Accordingly, new methods are needed for producing a Dkk1 protein with similar biological activity to the native form, in large quantities that was not costly. More particularly, there is a need to produce large quantities of Dkk, preferably Dkk1, to be used in high throughput screening (HTS) assays. This Dkk1 must be glycosylated in a pattern similar to the Dkk1 protein found in the host organism from which that Dkk1 derives. The loss of the glycosyl groups can significantly impact the activity of the protein in various assay systems.